Retroviruses interact intimately with their hosts, usurping cellular pathways to complete their life cycles. Identification of novel host factors that contribute to virus replication will enhance our understanding of retrovirus-cell interactions and may provide new targets for antiviral therapy. Host factors are known to play a major role in the late stages of virus particle formation and budding, but the early steps of assembly have remained understudied largely because distinct molecular events have been difficult to identify. Recently, we discovered an early step in Rous sarcoma virus (RSV) assembly: nucleocytoplasmic shuttling of the Gag protein, the viral factor that directs the assembly process. This nuclear trafficking event is crucial for the formation of infectious virus particles, and mutants that bypass the nuclear compartment incorporate reduced amounts of viral genomic RNA. We hypothesize that RSV genome recognition and selection, one of the earliest steps in particle assembly, occurs within the nucleus. This idea challenges the current dogma that retroviruses package their genomes in the cytoplasm; thus this research project has the potential to be paradigm-shifting. Our specific aims are centered on this novel hypothesis and address mechanistic aspects of nuclear shuttling of RSV Gag. We have a well-defined model system and an informative collection of Gag mutants with altered nuclear transport properties to use as tools. In Aim1, we will dissect the nuclear import pathway, define the roles of specific importins, and use inhibitors of entry to investigate the biological role of Gag nuclear trafficking in virus replication. Aim 2 focuses on intranuclear interactions between Gag proteins, Gag-RNA complexes, nucleolar factors, and subnuclear bodies. In Aim 3, we will utilize innovative imaging methods to visualize the movement of Gag-RNA complexes within the nucleus and from the nuclear envelope to the plasma membrane, identifying additional host factors involved in Gag- RNA trafficking. We will also be looking for evidence that Gag nuclear shuttling influences basic cellular processes, as many viruses have recently been shown to compromise nuclear export, activities of import factors, and localization of essential nuclear/nucleolar proteins. Through this focused experimental plan, we hope to shed light on fundamental aspects of retrovirus assembly, genome packaging, and nuclear events engaged in the interplay between retroviral pathogens and their host cells. PUBLIC HEALTH RELEVANCE: Retroviruses cause cancer and immunodeficiency syndromes in people and animals. This application focuses on defining the intracellular trafficking of the retroviral Gag protein, the major structural protein of retroviruses. Results of this research may enhance our understanding of how retroviruses interact with the cells they infect, potentially leading to new antiviral targets and enhanced retroviral vectors for gene therapy.